Trichohyalin (THH) is a major differentiation product of the inner root sheath cells of the hair follicle, the medulla of the hair fiber, and it is expressed in the epidermis and a variety of other hardened stratified squamous epithelial cells and tissues. Initially, we proposed that it functions at least in part as a keratin intermediate filament associated protein in these tissues. Of particular interest to us are the observations that THH is a substrate for transglutaminases (TGases), which cross-link it into polymers, and for the peptidylarginine deiminase (PAD) enzymes which convert protein-bound arginines to citrullines. The overall purpose of our studies are to understand the expression of the THH gene, the likely unique structure of THH protein, the way in which it is utilized in tissues such as the inner root sheath, and the details and consequences of these two major postsynthetic modifications of it. Because full-length human THH is very insoluble (<1 microgram/ml), we have expressed in bacteria domain 8 (about 40%) of THH (named THH-8) for use in the study of these two postsynthetic modification events. This product contains numerous peptide repeats which are typical of the intact THH, is highly a-helical (>90%), and its solubility is about 50 microgram/ml so that it is suitable for in vitro biochemical assays. We found that a commercially available PAD enzyme converts 60% of the arginines of THH-8 to citrullines, with a concomitant significant increase in solubility. Moreover, by circular dichroism, this results in the complete loss of the a-helical structure of THH-8. THH-8 is used by all three TGases known to be present in the epidermis as a complete substrate, that is, the THH-8 provides both the glutamine donor and lysine acceptor residues. However, by calculation of kinetic parameters, the TGase 3 enzyme uses it most efficiently. About 10% of the glutamines are used for crosslinking with high specificity to most of the lysines. Furthermore, the kinetic efficiency of the TGase 3 enzyme is greatly increased following maximal PAD modification. In this case, virtually all of the glutamines may be partially used for crosslinking to all of the lysines: that is, THH-8, and by inference intact THH in cells, becomes a more efficient substrate following denaturation. However, three additional members of the TGase family have been recently discovered. Each of these will now be tested using THH-8 as the substrate, in order to explore what role, if any, these new enzymes have in the processing of THH in vivo. Nevertheless, extant data suggest a model for the temporal order of the postsynthetic modifications of THH which we have explored in mouse hair follicles. Using indirect immunofluorescence procedures with specific antibodies, we found that THH expression precedes expression of the TGase 3 enzyme. Other data have shown that the PAD enzymes are expressed after the initiation of THH expression, but before TGase 3 expression. Therefore in the hair follicle, we propose that THH is first modified by PAD enzymes, which denature it and render it more soluble. Then the solubilized modified THH becomes a very efficient substrate for the TGase 3 enzyme which thereby cross-links it to a highly insoluble complex. We propose that additional lysines may be recruited for cross-linking, including those from the keratin intermediate filament of these THH-containing epithelia. This is especially important in the inner root sheath cells of the hair follicle, which contain about two-thirds keratin filaments and one-third THH. In this way, we propose that the THH effectively functions as an interfilamentous matrix protein to strengthen and harden the tissue. We are currently testing this hypothesis by performing sequencing experiments on peptides recovered from mouse inner root sheath and medulla tissues. The sequences of the mouse THH and keratin keratins of these tissues are now known. By identifying which proteins are cross-linked together, we can obtain for the first time detailed information on exactly how the THH is used and functions in these cell types. Such information may also aid in understanding the precise role of it in other tissues. THH contains the highest content of charged residues of any protein known in biology. In particular, it is proposed that the characteristic a-helical structure of native THH is stabilized by the formation of >1 salt bridges/3.5 residues/turn of the a-helix: that is, THH may be the only known example of a protein which can form a stable single-stranded a-helical conformation. We have made a 55-residue synthetic peptide that shares the conserved structural motifs of intact THH. This has been crystallized and good X-ray diffraction data have been obtained. A solved structure is imminent. The proximal promoter regions of the human and mouse THH genes encompass the first 160-175 bp above the transcription start site. This region contains an essential AP1 site as well as overlapping ets-like, NF-KB, Cre-like and Sp1-like sites, as well as some as yet unknown repressor elements. The functionality and synergistic interaction of these sites have been tested in transient expression of CAT constructs into hair follicles harvested from <3 day-old neonatal mice. These regions confer a high degree of specificity of expression in hair follicle tissue since they have no or only very low levels of expression in cultured epidermal keratinocytes of other epitheliod cell types. However, the mouse and human sequences and motifs are quite different. Nevertheless, these various elements operate synergistically to confer tissue specific expression. Thus further work is in progress to explore the details of these synergistic interactions. We are particularly interested in defining those element(s) responsible for hair-follicle specificity, as such motifs will be of great value in the study of other hair follicle-expressed genes.